EP2588863B1 - Erfassung von mikroorganismen - Google Patents

Erfassung von mikroorganismen Download PDF

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Publication number
EP2588863B1
EP2588863B1 EP11734173.5A EP11734173A EP2588863B1 EP 2588863 B1 EP2588863 B1 EP 2588863B1 EP 11734173 A EP11734173 A EP 11734173A EP 2588863 B1 EP2588863 B1 EP 2588863B1
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EP
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Prior art keywords
beads
bacteria
fungi
fragments
solid surface
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English (en)
French (fr)
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EP2588863A1 (de
Inventor
Christopher John Stanley
Stuart Mark Wilson
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MICROSENS DIAGNOSTICS Ltd
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Microsens Diagnostics Ltd
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/14—Streptococcus; Staphylococcus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/10—Enterobacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)

Definitions

  • the present invention relates to methods of capturing bacteria and fungi from suspension, and also to their subsequent detection and/or identification.
  • micro-organisms may initially be present in a large volume sample material, such as sputum, blood, dispersed food or potable water. Concentration into a small volume that is free of sample contaminants optimises sensitivity and removes inhibitors in diagnostic procedures, such as microscopy, immunoassay or nucleic acid amplifications processes such as PCR.
  • the capture conditions may not be suitable for an antibody-based system: for example, in the capture of mycobacteria from sputum that has been thinned with sodium hydroxide and which remains at a high pH which would inactivate antibodies. In these and other circumstances of harsh conditions antibody capture cannot be used.
  • blood products such as in platelet screening, it is important to know if the blood products (in this case platelets) are contaminated with any microorganism at all. Under such circumstances it would be hard to do this with an antibody-based approach as antibodies tend to be organism or species-specific.
  • JP2001112497 describes removing Mycobacteria from an alkali decontaminated liquid by precipitating calcium phosphate therein, the precipitate presumably carrying down the bacteria as it forms.
  • WO 2009/086343 describes the use of a carbohydrate-coated surface together with a biotin binding protein and an amphiphilic glycoside of a steroid or triterpene to bind microorganisms.
  • WO28072242A2 describes a polymeric matrix derivatised with amino acids as a binding surface and WO29046191A2 describes the use of diatomaceous earth particles coated with various metallic or inorganic surfaces to achieve the same result.
  • WO95/32304 uses an aqueous two-phase polymer separation system to selectively enhance the number of target micro-organisms with respect to background flora, but does not suggest capture to a solid surface.
  • EP0988307 discloses a method of isolating nucleic acid from a sample of cells, said method comprising: (a) binding cells in said sample to a solid support to isolate cells from the sample; (b) lysing the isolated cells; and (c) binding nucleic acid released from said lysed cells to said same solid support.
  • WO2008/065047 discloses a method for the capture from a sample of micro-organisms having a hydrophobic surface such as mycobacteria, comprising contacting the micro-organisms with a capture reagent which has both a hydrophobic character to bind the micro-organisms by hydrophobic interaction and a polar character, so as to capture the micro-organisms to an acidic surface.
  • WO2007/068904 discloses a method for detecting the presence or absence of a target cell in a sample, said method comprising (a) binding cells in said sample to a particulate and mixable solid support; (b) eluting the cells from the solid support without the use of competitor molecules to disrupt the interaction between the cell and the solid support; (c) after lysis of said cells, detecting the presence or absence of nucleic acid characteristic of said target cell, wherein said solid support does not have antibodies or antibody fragments immobilised thereon.
  • the present invention accordingly now provides a method of capturing onto a solid surface bacteria and fungi and/or fragments thereof in an aqueous liquid, comprising adding to said liquid a sufficient quantity of a water soluble polymer in the presence of said solid surface to displace said bacteria and fungi and/or fragments thereof from the liquid to the solid surface, wherein the bacteria and fungi are Mycobacteria or fragments thereof, and/or the solid surface is provided by (a) paramagnetic beads with a carboxy surface or (b) a polymer having negatively charged surface groups.
  • the process may comprise providing together a solid surface and an aqueous liquid containing bacteria and fungi and/or fragments thereof and a water soluble polymer so as to displace said bacteria and fungi and/or fragments thereof from said liquid onto said solid surface, wherein the bacteria and fungi are Mycobacteria or fragments thereof, and/or the solid surface is provided by (a) paramagnetic beads with a carboxy surface or (b) a polymer having negatively charged surface groups.
  • Fragments of bacteria and fungi which can be captured in this way include sedimentable fragments that could be centrifuged to deposit from the liquid using centrifugation at 4,000 x g for 20 min which is the speed that is normally used to sediment bacteria.
  • the method also works to capture fragments, which may include soluble proteins or other soluble cell components, that cannot be caused to deposit under such centrifugation conditions and which may be termed non-sedimentable.
  • the concentration of the added water soluble polymer in the aqueous liquid may be freely adjusted according to the nature of the polymer so as to produce the desired displacement of the bacteria and fungi and/or fragments from suspension or solution in the liquid to be deposited on the solid surface.
  • the polymer concentration is from 2 to 30% w/v, preferably 5 to 20% w/v, more preferably 7 to 15% w/v, most preferably about 10% w/v. These concentrations are particularly suitable for use with PEG or polyvinylpyrrolidone (PVP) as the polymer.
  • the water soluble polymer is a non-ionic hydrophilic polymer. It may for instance be a dextran, a PVP or a PEG.
  • the PEG may be polydisperse in molecular weight or monodisperse, may be branched or straight chain and may be of a star type.
  • the water soluble polymer has a weight average molecular weight of from 200 to 100,000. It may for instance have a molecular weight of from 1,000 to 20,000, more preferably 5,000 to 13,000, e.g. about 8000. These molecular weights are particularly suitable in the case of PEG or PVP.
  • Binding of the bacteria and fungi to the solid surface may be enhanced by the use of high ionic strength conditions.
  • the ionic strength of the aqueous liquid is raised by the addition of a water soluble inorganic salt to from 150mM to 6M, more preferably from 0.25 to 0.75M, e.g. 0.5M.
  • salts can be added to the solution to achieve a high ionic strength, for example, ammonium sulphate, ammonium chloride, magnesium sulphate, magnesium chloride but sodium chloride is preferred.
  • the binding to a surface can be performed in conditions of high or low pH in the range 1-14.
  • a high pH such as from 9 to 14 may be preferred in order to kill or reduce the ability to reproduce of other kinds of bacteria that may be present.
  • the binding to a surface can be performed in the presence of a detergent which can include ionic (such as sodium dodecylsulphate) or non-ionic (such as Tween20 and Triton X100) detergents.
  • a detergent which can include ionic (such as sodium dodecylsulphate) or non-ionic (such as Tween20 and Triton X100) detergents.
  • the solid surface has a high specific surface area and hence, preferably the solid surface is provided by beads.
  • the term 'beads' includes but is not limited to insoluble particles that are spherical or irregular in nature and of size ranging from 0.1 micron to 5 mm in size.
  • a method according to the invention may further comprise separating said solid surface from said liquid.
  • centrifugation can be used to concentrate whole organisms but fragments of organisms may be non-sedimentable at the speed and time used.
  • centrifugation requires an instrument and also this step cannot easily be incorporated into an automated procedure for microorganism concentration and detection.
  • after centrifugation it is difficult to resuspend reliably the sedimented pellet containing the microorganisms in the desired small volume, often larger volumes are required to ensure full dispersal of the pellet in the tube.
  • the nature of the solid surface on which the bacteria and fungi and/or fragments are received is such that the solid surface can be separated from the suspending liquid without the use of filtration or centrifugation. It is therefore preferred that the beads are attractable to a magnet for separation or are sufficiently dense that they can be separated by sedimentation even if the polymer containing liquid is quite viscous. Therefore, it is preferred that said beads are paramagnetic or have a density sufficiently above the liquid that they sediment without centrifugation, e.g. above 2.0g/ml, more preferably above 3.0g/ml. Alternatively, they may have a density sufficiently below that of the liquid that they will separate to float on the liquid, e.g. hollow beads, which may be of glass. Such buoyant beads may have a density below 0.8g/ml, e.g. from 0.4 to 0.7mg/ml.
  • Suitable dense bead materials, or bead core materials are aluminium oxide, silicon carbide, silicon nitride, titanium carbide, iron oxide (such as magnetite) and glass.
  • the solid surface may be of a polymer having positively charged or negatively charged surface groups.
  • suitable surface groups are amine, quaternary ammonium, carboxylic acid, sulphonic acid, or sulphate groups.
  • Suitable materials include dextran sulphate and poly(diallyldimethylammonium chloride) (pDADMAC).
  • the bead may be of one material throughout or may have a core bearing a surface coating.
  • particles presenting a surface of magnetite, uncoated magnetite particles has particular advantages. Such particles are ferromagnetic (in the broad sense, including ferrimagnetic) and so are readily separated from liquid medium and bacteria and fungi and/or fragments thereof are readily caused to adhere to the surface of such particles by the presence in liquid medium of soluble polymers. Moreover, the removal of liquid medium containing such polymers and its replacement by a liquid in which such polymers are not present provides a ready means of removing the captured materials from the solid surface.
  • a method according to the invention may further comprise eluting said bacteria and fungi and/or fragments from said solid surface after separation thereof from said liquid.
  • the elution can be performed under a wide range of conditions, for instance in buffers with a range of salt concentrations from 150 mM to 6 M.
  • non-ionic, ionic or zwitterionic detergents such as Tween20, Triton X-100, CTAB, sarkosyl or SDS can be included to aid elution from the bead surface.
  • the bacteria and fungi and/or fragments are eluted from the solid surface into a volume of liquid which is less than the volume of the liquid in which the bacteria and fungi and/or fragments were originally suspended by a factor of at least 2, e.g. at least 4.
  • a method of the invention may further comprise detecting the bacteria and fungi and/or fragments which were captured onto the solid surface. This may be performed after elution from the solid surface or whilst the bacteria and fungi and/or fragments remain on the solid surface. It may be performed either after removing the aqueous liquid from the solid surface or whilst it still remains. For instance, after capturing the bacteria and fungi and/or fragments to beads, a labelled binding agent such as an antibody specific for the bacteria and fungi and/or fragments or auramine stain may be added and the liquid may be run through a cell sorter apparatus to detect labelled beads.
  • a labelled binding agent such as an antibody specific for the bacteria and fungi and/or fragments or auramine stain may be added and the liquid may be run through a cell sorter apparatus to detect labelled beads.
  • Beads or other forms of solid surface bearing bacteria and fungi and/or fragments may be stained with a bacteria and fungi stain, either directly or after culturing the organism to increase the number thereof.
  • Mycobacteria may be revealed by the use of an acid fast stain for visualisation.
  • Other forms of detection such as nucleic acid amplification techniques (e.g. PCR), detection of metabolites, antibody based detections such as ELISA for the bacteria and fungi themselves or cell components thereof may be used.
  • One suitable ELISA method for detecting Mycobacteria and/or fragments is for instance disclosed in GB1004710.8 . Capture methods described herein may be used as modifications of the methods described in GB1004709.0 . Detection methods used may be of the kind that require the presence of viable bacteria and fungi, such as culturing.
  • Example 1 Demonstration of organisms binding to different bead types.
  • the E. coli was concentrated using carboxy paramagnetic beads (Invitrogen) in buffers containing different combinations of PEG and NaCl.
  • Binding buffer used Extent of capture (%) 0.5 M NaCl 0 0.5 M NaCl, 10% (w/v) PEG 8000 100 10% (w/v) PEG 8000 25
  • Bacteria in general are negatively charged and would be repelled by the negatively charged carboxy beads. Such repulsion may aid removal of the bacteria from the beads at a later stage.
  • PEG NaCl only
  • PEG alone there is no binding of the E. coli to the beads.
  • PEG alone there is some binding but the efficiency is increased by the presence of NaCl which presumably helps to sequester the negative charges both on the bacilli and on the beads and thus promotes bacteria binding to the beads.
  • Example 3 Concentration of Mycobacterium tuberculosis from sputum using paramagnetic beads.
  • the Mycobacterium tuberculoses was concentrated from thinned sputum which has a high pH using beads (BioMag Amine beads, Bangs Laboratories, catalogue number BM546) coated with poly diallyl dimethyl ammonium chloride (pDADMAC). The presence of TB on the beads was then confirmed by acid fast microscopy.
  • Example 4 Concentration of Mycobacterium tuberculosis from sputum using settling beads.
  • the Mycobacterium tuberculosis (TB) is concentrated from thinned sputum which has a high pH using 50 micron silicon carbide beads either uncoated or coated with poly diallyl dimethyl ammonium chloride (pDADMAC). The capture of TB was then confirmed by acid fast microscopy.
  • pDADMAC poly diallyl dimethyl ammonium chloride
  • mycobacteria can be captured from thinned sputum at high pH using coated or uncoated silicon carbide beads and high concentrations of PEG.
  • the Mycobacterium tuberculosis is concentrated from the sputum in a two-step procedure.
  • the TB is concentrated by centrifugation and the pellet from this step is captured onto beads in the presence of high salt and PEG8000.
  • the presence of TB on the beads is then confirmed by acid fast microscopy.
  • This example demonstrates the two step concentration of TB from sputum.
  • the first step involved centrifugation which concentrated the TB into a 1 ml volume. This volume was too large to be applied directly to a microscope slide so the TB was further concentrated by bead capture to a volume that was small enough to be applied to a slide for microscopy.
  • Example 6 Concentration of Mycobacterium tuberculosis from sputum under different bead coating and binding conditions.
  • polybreen-coated beads can be used to capture mycobacteria from sputum.
  • the silicon carbide beads in the presence of 10% PEG were able to capture and concentrate the non-sedimentable fragments of mycobacteria that could then be detected by ELISA.
  • Example 8 Concentration of Mycobacterium tuberculosis from sputum using different polymers and magnetic beads composed of magnetite Rationale
  • Polyvinylpyrrolidone of average molecular weight 10,000 Polyethylene glycol of average molecular weight 200
  • Polyethylene glycol of average molecular weight 8000 All supplied by Sigma Aldrich, USA.
  • the PEG/magnetite extraction system can extract bacteria, fungi and soluble bacterial components. In this experiment we show that it can also extract viruses.
  • the Adenovirus was captured from solution by the combination of PEG8000 and magnetite and was retained on the bead through two washes prior to elution and detection by PCR.
  • mycobacteria and fungi from large sample volumes that may include harsh conditions of pH or salt concentrations we have developed an improved method that involves the use of polyethylene glycol and similar substances to deposit the bacteria and fungi or fragments onto the surface of a capture matrix.
  • polyethylene glycol removes the dependency of capture on the nature of the surface of the capture matrix and we have shown that many different capture matrices of many different surface natures can be used to capture a range of bacteria and fungi.
  • the beads can be paramagnetic, ferromagnetic (including ferrimagnetic), or simple silicon carbide or aluminium oxide but particularly useful embodiments for most bacilli and fungi use paramagnetic beads with a carboxy surface, or uncoated silicon carbide beads, or uncoated magnetite beads.
  • the bacteria and fungi can be concentrated using a magnet to remove the beads from suspension and then eluted in a small volume of buffer with reduced or no polyethylene glycol content.
  • the bacteria and fungi can be concentrated and eluted, after bead settling, in a small volume of buffer with reduced or no polyethylene glycol content.

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  • Urology & Nephrology (AREA)
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  • Tropical Medicine & Parasitology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
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Claims (15)

  1. Verfahren zum Einfangen von Bakterien und Pilzen und/oder Fragmenten von Bakterien und Pilzen, die in einer wässrigen Flüssigkeit vorliegen, auf einer festen Oberfläche, wobei das Verfahren das Zugeben einer ausreichenden Menge eines wasserlöslichen Polymers zu der Flüssigkeit in Gegenwart der festen Oberfläche umfasst, um die Bakterien und die Pilze und/oder die Fragmente davon aus der Flüssigkeit auf die feste Oberfläche zu treiben, wobei die Bakterien und die Pilze Mykobakterien oder Fragmente davon sind und/oder die feste Oberfläche durch (a) paramagnetische Kügelchen mit einer Carboxyoberfläche oder (b) ein Polymer mit negativ geladenen Oberflächengruppen bereitgestellt wird.
  2. Verfahren nach Anspruch 1, wobei die Konzentration des zugegebenen wasserlöslichen Polymers in der wässrigen Flüssigkeit 2 bis 40 Gew.-%/Vol.-% beträgt.
  3. Verfahren nach Anspruch 2, wobei die Konzentration 5-15 Gew.-%/Vol.-% beträgt.
  4. Verfahren nach einem vorhergehenden Anspruch, wobei das wasserlösliche Polymer ein nichtionisches hydrophiles Polymer ist.
  5. Verfahren nach einem vorhergehenden Anspruch, wobei das wasserlösliche Polymer ein Dextran, PVP oder PEG ist.
  6. Verfahren nach einem vorhergehenden Anspruch, wobei das wasserlösliche Polymer ein Molekulargewicht von 1000 bis 20.000 aufweist.
  7. Verfahren nach einem vorhergehenden Anspruch, wobei die Ionenstärke der wässrigen Flüssigkeit durch die Zugabe eines wasserlöslichen anorganischen Salzes auf 150 mM bis 6 M erhöht wird.
  8. Verfahren nach einem vorhergehenden Anspruch, wobei die Salzkonzentration 0,25 bis 2,5 M beträgt.
  9. Verfahren nach einem vorhergehenden Anspruch, wobei die feste Oberfläche durch Kügelchen bereitgestellt wird.
  10. Verfahren nach Anspruch 9, wobei die Kügelchen paramagnetisch oder ferromagnetisch sind oder eine derartige Dichte aufweisen, dass sie sich beim Stehenlassen separieren.
  11. Verfahren nach Anspruch 10, wobei die Kügelchen aus Magnetit oder Siliciumcarbid sind.
  12. Verfahren nach einem vorhergehenden Anspruch, wobei die feste Oberfläche aus einem Polymer mit positiv geladenen oder negativ geladenen Oberflächengruppen ist.
  13. Verfahren nach Anspruch 12, wobei die Oberflächengruppen, Amin-, quartäre Ammonium-, Carbonsäure-, Sulfonsäure oder Sulfatgruppen sind.
  14. Verfahren nach einem vorhergehenden Anspruch, das weiterhin das Trennen der festen Oberfläche von der Flüssigkeit umfasst und weiterhin das Eluieren der Bakterien und der Pilze und/oder der Fragmente davon von der festen Oberfläche nach der Trennung dieser von der Flüssigkeit in ein Flüssigkeitsvolumen umfasst, das um einen Faktor von mindestens 2 kleiner als das Volumen der Flüssigkeit ist, in dem die Bakterien und die Pilze und/oder die Fragmente davon ursprünglich suspendiert wurden.
  15. Verfahren nach einem vorhergehenden Anspruch, das weiterhin das Nachweisen der Bakterien und der Pilze und/oder der Fragmente davon umfasst, die auf der festen Oberfläche eingefangen wurden.
EP11734173.5A 2010-07-02 2011-06-29 Erfassung von mikroorganismen Active EP2588863B1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB1011152.4A GB201011152D0 (en) 2010-07-02 2010-07-02 Capture of micro-organisms
PCT/GB2011/051225 WO2012001407A1 (en) 2010-07-02 2011-06-29 Capture of micro-organisms

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EP2588863A1 EP2588863A1 (de) 2013-05-08
EP2588863B1 true EP2588863B1 (de) 2017-07-19

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US (1) US8603771B2 (de)
EP (1) EP2588863B1 (de)
CN (1) CN103189747B (de)
AU (1) AU2011273142B2 (de)
CA (1) CA2806468C (de)
GB (1) GB201011152D0 (de)
RU (1) RU2573921C2 (de)
WO (1) WO2012001407A1 (de)
ZA (1) ZA201300826B (de)

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WO2016018475A1 (en) 2014-07-31 2016-02-04 Kimberly-Clark Worldwide, Inc. Anti-adherent composition
MX384467B (es) 2015-04-01 2025-03-14 Kimberly Clark Co Sustrato fibroso para la captura de bacterias gramnegativas.
WO2017042819A1 (en) 2015-09-11 2017-03-16 Molecular Detection Israel Ltd. Methods for isolating microbial cells from a blood sample
MX2018008569A (es) 2016-01-28 2018-08-16 Kimberly Clark Co Composicion antiadherente contra virus de acido desoxirribonucleico (adn) y metodo para inhibir la adherencia de virus de acido desoxirribonucleico (adn) a una superficie.
US11168287B2 (en) 2016-05-26 2021-11-09 Kimberly-Clark Worldwide, Inc. Anti-adherent compositions and methods of inhibiting the adherence of microbes to a surface
CN112574885A (zh) * 2019-09-29 2021-03-30 广东体必康生物科技有限公司 一种基于磁珠富集痰液样本的结核分枝杆菌检测方法及其专用试剂盒
CN111961706A (zh) * 2020-08-13 2020-11-20 恺硕生物科技(厦门)有限公司 一种核酸提取纯化方法
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RU2573921C2 (ru) 2016-01-27
AU2011273142A1 (en) 2013-02-21
ZA201300826B (en) 2014-04-30
EP2588863A1 (de) 2013-05-08
GB201011152D0 (en) 2010-08-18
US8603771B2 (en) 2013-12-10
CN103189747B (zh) 2015-03-11
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AU2011273142B2 (en) 2015-09-17
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